Neuronal replacement and reestablishment of axonal connections

Abstract

The present invention provides compositions and methods for modulation of neuronal networks in the CNS and/or PNS. In certain embodiments, the invention includes modulation of existing networks or restoring one or more damaged or lost axonal connections. In one embodiment, the invention comprises a tissue-engineered composition comprising an elongated tubular construct having at least one neuron and axon extending through the core of the construct.

1 - 19 . (canceled) 
     
     
         20 . A method of culturing and implanting a micro-tissue engineered neural network in a mammal to promote structural integration of the micro-tissue engineered neural network with the host brain tissue of the mammal, the method comprising:
 providing an elongated tubular hydrogel construct having a first end and a second end, the construct comprising a tubular body comprising at least one biopolymer and having an inner surface defining a luminal core comprising at least one extracellular matrix protein;   positioning a plurality of neuronal progenitor cells at the first end of the construct;   culturing the plurality of neuronal progenitor cells at the first end of the construct in vitro to:
 (i) mature the neuronal progenitor cells into a plurality of neurons comprising one or more differentiated neuronal subtypes selected from the group consisting of glutamatergic neurons, GABAergic neurons, and dopaminergic neurons; and 
 (ii) promote growth of axons of the plurality of neurons through at least a portion of the luminal core, along a length of the construct, thereby forming the micro-tissue engineered neural network comprising the construct and neurons having an engineered cytoarchitecture, such that soma of the neurons are located at the first end and the grown axons extend through the core, and run longitudinally for at least a portion of a length of construct; and 
   implanting the micro-tissue-engineered neural network into the mammal.   
     
     
         21 . The method of  claim 20 , wherein the mammal is a rat. 
     
     
         22 . The method of  claim 20 , wherein the at least one biopolymer is at least one selected from the group consisting of hyaluronan, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin and agarose. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 20 , wherein the construct has an outer diameter from about 500 μm to about 1 mm. 
     
     
         26 . The method of  claim 20 , wherein the construct has an inner diameter from about 125 μm to about 500 μm. 
     
     
         27 . The method of  claim 20 , wherein the length of the construct is from about 0.1 mm to about 10 cm. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 20 , wherein the neuronal progenitor cells are obtained from rat. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 20 , wherein the method restores an axonal connection in the peripheral nervous system (PNS). 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 20 , wherein implanting the micro-tissue-engineered neural network comprises loading the micro-tissue-engineered neural network into a needle and injecting the micro-tissue-engineered neural network into a tissue of the mammal. 
     
     
         46 . The method of  claim 20 , wherein implanting the micro-tissue-engineered neural network comprises directly penetrating a tissue of the mammal with the micro-tissue-engineered neural network. 
     
     
         47 . The method of  claim 20 , wherein the extracellular matrix protein is selected from the group consisting of collagen, fibronectin, fibrin, hyaluronic acid, elastin, and laminin. 
     
     
         48 . The method of  claim 20 , wherein the construct comprises a protective outer coating on an outer surface of the tubular body, the protective outer coating comprising carboxymethylcellulose. 
     
     
         49 . A method of promoting neurite outgrowth in a rat, the method comprising:
 providing a micro-tissue-engineered neural network, wherein:
 the micro-tissue-engineered neural network comprises an elongated tubular hydrogel construct; wherein the tubular hydrogel construct having a first end and a second end, the construct comprising a tubular body comprising an agarose-extracellular matrix hydrogel, wherein the hydrogel comprises 1-4% agarose in phosphate-buffered saline; and having an inner surface defining a luminal core comprising salmon-derived fibrin matrix or a blend of collagen and laminin; and having an outer surface having a coating comprising carboxymethylcellulose; 
   positioning a plurality of cortical neurons isolated from rats at embryonic day 18, wherein the cortical neurons are positioned at the first end of the construct, and further wherein the plurality of cortical neurons are at a density of 56000, 72000 or 36000 cell/ml;   culturing the plurality of cortical neurons at the first end of the construct; and   implanting the micro-tissue-engineered neural network by stereotaxically injecting the micro-tissue-engineered neural network into the cortex in the rat.   
     
     
         50 . A method of promoting neurite outgrowth in a rat, the method comprising:
 providing a micro-tissue-engineered neural network, wherein:
 the micro-tissue-engineered neural network comprises an elongated tubular hydrogel construct; wherein the tubular hydrogel construct comprises a first end and a second end, the construct comprising a tubular body comprising an agarose-extracellular matrix hydrogel, wherein the hydrogel comprises 1-4% agarose in phosphate-buffered saline; and an inner surface defining a luminal core comprising collagen; and having an outer surface having a coating comprising carboxymethylcellulose; 
   positioning a plurality of dorsal root ganglia (DRG) neurons isolated from rats at embryonic day 16, wherein the DRG neurons are positioned at the first end of the construct;   culturing the plurality of DRG neurons at the first end of the construct; and   implanting the micro-tissue-engineered neural network by stereotaxically injecting the micro-tissue-engineered neural network into the cortex in the rat.